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Azam M. Mansoor, Tonunn Fiskerstrand, Per M. Ueland, and Helga Refsum ...... Allen. RH, Stabler SP, Savage DG, Lindenbauin J. Diagnosis of colsilamin.
CUN. CHEM. 39/7,

1390-1397

(1993)

Kinetics of Plasma Homocysteine in Healthy Subjects after Peroral Homocysteine Loading Anne Bent

Guttormsen,’

Azam

M. Mansoor,

Tonunn Fiskerstrand,

The kinetics of plasma homocysteine were determined in 13 healthy subjects after peroral administration and in one person after intravenous injection. Various forms of homocysteine completely dissolved in an aqueous solution were rapidly absorbed after peroral administration, and the bioavailability was estimated to be 0.53. The volume of distributionwas 0.66 L/kg. The area under the plasma concentration curve (AUCQ h) was proportional to the administered dose (33.5-134 moI/kg body wt), and showed small interindividualvariations. Plasma homocysteine showed first-order elimination kinetics for at least 6 h. The half-life (t112)was 223 ± 45 mm, and there was a significant correlation between t112values determined on two different occasions in the same individual. The transient hyperhomocysteinemia was associated with an increase in plasma methionine, which probably reflects intracellular remethylation of homocysteine. Less than 2% of the administered homocysteine dose was recovered in the urine. These findings may form the basis for future studies on the regulation of plasma homocysteine in health and disease, and should motivate the evaluation of a homocysteine loading test as a diagnostic tool. Indexing

Terms: amino acids

.

metabolism

.

methlonine

urine Homocysteine

is a sulfur

amino

acid formed

from

methionine as a product of transmethylation. Intracellular homocysteine is either salvaged to methionine or degraded to cysteine. The former reaction is in most tissues catalyzed by the enzyme 5-methyltetrahydrofolate-homocysteine S-methyltransferase (methionine synthase; EC 2.1.1.13), which requires 5-methyltetrahydrofolate as methyl donor and cobsilamun as cofactor. The first step in homocyst.eine degradation is catalyzed by the vitamin B6-dependent enzyme cystathionine -synthase (1). Homocysteine export into the extracellular medium is an alternative route of homocysteine disposal and, under conditions of impaired metabolism, the extracellular homocysteine concentration, like that in plasma and urine, becomes markedly increased (2). The export into the extracellular medium is the biochemical basis for plasma homocysteine as a marker for several disease states (2). Plasma homocysteine is markedly increased during folate (3) or cobelamin deficiency (4). It is also increased to high amounts in patients with the rare inborn error

Per M. Ueland, and Helga Refsum

called homocystinuria, which is most commonly caused by deficiency of the enzyme cystathionine -synthase. These patients have a high morbidity of cardiovascular disease, which may cause death in early adolescence and even in childhood (5). Recent clinical studies including more than 1800 patients have amply demonstrated that a moderate increase of plasma homocysteine, socalled hyperhomocysteinemia, is a common and independent risk factor of premature cardiovascular disease in the general population. Hyperhomocysteinemia is caused by both nutritional and genetic factors, including heterozygosity for cystathionine p-synthase deficiency (6, 7). The plasma homocysteine concentration in fasting subjects heterozygous for cystathiomne fl-synthase deficiency is usually within the normal range. To identify such subjects, the methionine loading test has been used in several clinical studies (6, 7). The test is based on peroral administration of a standard dose of methionine and measurement of the plasma homocysteine concentration 4-6 h after intake. The test improved the dis-

of heterozygotes from controls in some studies, but discontinuous segregation between the two groups was not obtained (8). This may reflect the interindividual variation in response to a standard methionine load (9), which may be due to, among other factors (9), the fact that plasma homocysteine is a function of both homocysteine formation and metabolism. Homocystine loading has been attempted in patients with homocystinuria, but the results were not interpretable because of the low solubility and absorption of this compound (10). In another attempt, free base of racemic DL-homocysteine was given to eight homocystinurics and four healthy subjects, and the increase in the free homocysteine mixed and symmetric disulfides was measured in plasma (11). No data exist on the plasma kinetics of total homocysteine in normal subjects given peroral doses of L-homocysteine. Such data may provide information of homocysteine disposition and metabolism in healthy subjects. In addicrimination

tion, the kinetic features should be decisive regarding further evaluation of a homocysteine loading as a test to reveal defects in homocysteine metabolism. In the present work we characterized the plasma kinetics of L-homocystame in 13 healthy subjects.

Materials and Methods Materials and Subjects

Department of Pharmacology and Toxicology, Armauer Hansens Hus, University of Bergen, N-5021 Haukeland Hospital, Bergen, Norway. 1 Author for correspondence. Received December 9, 1992; acceptedFebruary 11, 1993. 1390 CUNICAL CHEMISTRY, Vol. 39, No. 7,

1993

Sources of most reagents used have been given previously (12). L-Homocysteine thiolactone and L-homocystine were purchased from Sigma Chemical Co., St. Louis,

MO.

Thirteen healthy volunteers, six women and seven men (ages 19-36 years), participated in the study. The participants had provided their written informed consent, and the protocol was approved by the regional ethical committee of western Norway. All subjects had plasma concentrations of folate, coba1min, total homocysteine, total cysteine, and methionine within the normal range (Table 1). Homocyst(e)ine

Solutions

All homocyst(e)ine solutions ately before administration.

were prepared immedi-

Reduced homocysteine was otherwise stated. Powdered L-homocysteine thiolactone (300-1200 mg, 33.5134 molJkg of body wt) was dissolved in 5 mL of 5 mol/L NaOH and allowed to stand for 5 mm to open the thiolactone ring. We then added 5 mL of 5 mol/L HC1 for Peroral

administration.

the tIministered

neutralization,

form, unless

and carefully

adjusted the pH to be-

tween 4 and 5. A mixture of water and apple cider (to mask the unpleasant taste of homocysteine) was added, to give a total volume of 200 mL. L-Homocystine (526 mw, 67 mol/kg) and L-homocysteine thiolactone (600 mg; 67 molJkg) for peroral use were dissolved in 25 mL of 1 mol/L HC1 and in 25 mL of 1 mol/L NaC1, respectively. The former solution was adjusted to pH between 4 and 5 by adding 1 mol/L NaOH. Both solutions were diluted with water and apple cider to a final volume of 200 mL. Intravenous injection. Reduced homocysteine was

given in these experiments.

L-Homocysteine

thiolactone

(720 mg) was dissolved in 4.4 mL of 2.7 mol/L NaOH and allowed to stand for 5 mm. This solution was then carefully adjusted to pH 6 with HC1. Distilled

powder

water was added to give a volume of 60 mL and an osmolarity of about 500 mosmol/kg. The solution was sterilized

by ifitration (0.22-tm pore size), and 50 mL into the subject.

was injected

Table 1. CharacterIstics

Protocol Peroral loading. After an overnight fast, 13 persons received a standard peroral dose of 67 mol of reduced homocysteine per kilogram of body weight. This corresponds to a dose of L-homocysteine thiolactone of 10 mg/kg body weight. Ten of these subjects (five men and five women) were given a second loading. To obtain blood samples, we inserted a 1.7-mmdiameter cannula into a cubital vein. Immediately before the administration of homocysteine, blood was collected for determination of plasma folate and cobalamin and of preload values for the plasma aminothiols and methionine.

The homocysteine solution (200 mL) was swallowed in